Bacterial motility and its driving force, the flagellar motor, are important virulence factors of B. burgdorferi (the causative agent of Lyme disease) and many other bacteria. The flagellar motor is a remarkable nano-machine, powered by the proton (or sodium) gradient across the cytoplasmic membrane. The coupling of an electrochemical gradient to mechanical rotation is one of the most fascinating features of this molecular machine. The flagellar motor is composed of two major components: the stator and the rotor. Although prior structural studies have revealed the stunning complexity of the flagellar rotor, the mechanism of energy coupling in the flagellar motor remains poorly understood at the molecular level, mainly because of the lack of structural information about the membrane-bound stator and the rotor- stator interactions involved in flagellar rotation. The central hypothesis is that a high- resolution structure of the rotor-stator-C ring interface will provide vital structural information needed to propose models on the mechanisms of rotation and reversal. The objective of this application is to determine the structure/function relationship of the intact flagellar motor in situ by combining novel high throughput Cryo-Electron Tomography (Cryo-ET) approaches with genetic analysis to study the model system, B. burgdorferi. By collaborating with Drs. Steven Norris, Nyles Charon. MD Motaleb, Chunhao Li and Hanspeter Winkler, we propose to focus on two specific aims: Specific Aim 1 - Determine the detailed structure of the torque-generating unit by analyzing the 3-D structures of the complete flagellar motor and the purified flagellar rotor at 2 nm resolution. Specific Aim 2 - Determine the structural and functional roles of individual flagellar proteins by comparative analysis of wild-type organisms and flagellar gene mutants. We believe that the detailed analysis of the rotor/stator assembly in situ may provide the clearest avenue yet available to understanding of the mechanism of flagellar rotation and bacterial motility, which will in turn be applicable to the pathogenesis of all spirochetes and other motile bacteria. In addition, the further development of high-throughput Cryo-ET as part of this project will be readily applied to gain new insights into the structural and functional relationship of macromolecular machines related to pathogenesis of a variety of human pathogens, and offer a wide spectrum of important biomedical information at molecular resolution in living organisms.